Mycobacterium fortuitum strain

ABSTRACT

Novel compounds prepared by microbial transformation using novel mutants to selectively degrade steroids with or without 17-alkyl side chains of from 2 to 10 carbon atoms, inclusive. These compounds can be used as intermediates to make useful steroids.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a division of Ser. No. 877,230 filed Feb. 13, 1978 now U.S. Pat.No. 4,304,860 which is a continuation-in-part of application Ser. No.767,369, filed Feb. 10, 1977, now abandoned which is a division ofapplication Ser. No. 632,650, filed Nov. 17, 1975, which issued as U.S.Pat. No. 4,042,459.

BACKGROUND OF THE INVENTION

The transformation of steroids by microorganisms has been widely studiedand documented. Apparently, the earliest such work was by Mamoli andVercellone in 1937, Ber. 70, 470 and Ber. 70, 2079. They disclosed thereduction of 17-ketosteroids to 17β-hydroxysteroids by fermenting yeast.More recently, Peterson and Murray disclosed the 11α-hydroxylation ofprogesterone with the fungus Rhizopus nigricans: see, U.S. Pat. No.2,602,769 (1952). Also recently, Kraychy et al. in U.S. Pat. No.3,684,657 (1972) discloses the selective microbiological degradation ofsteroidal 17-alkyls by fermenting a steroid containing at least 8carbons in the 17-alkyl side chain with Mycobacterium sp. NRRL B-3683 toprepare androst-4-ene-3,17-dione, androst-1,4-diene-3,17-dione, and20α-hydroxymethyl-pregna-1,4-dien-3-one. Even more recently, Marsheck etal. in U.S. Pat. No. 3,759,791 (1973) disclose the selectivemicrobiological preparation of androst-4-ene-3,17-dione by fermenting asteroid of the cholestane or stigmastane series containing at least 8carbons in the 17-alkyl side chain with Mycobacterium sp. NRRL B-3805.

Compound III of the subject invention has been disclosed by Wang and Sihin 1963 (Biochem. 2: 1238-1243) as an intermediate in the degradation ofandrost-4-ene-17βol-3-one by Nocardia restrictus. The open ring form ofcompound I, 7α-methyl-perhydroindanedione-(1,5)-[β-propyl alcohol-(4)],has been disclosed by Schubert et al. in 1964 (Steroids 4: 581-586) asan intermediate in the degradation of progesterone by Mycobacteriumsmegmatis.

BRIEF SUMMARY OF THE INVENTION

Mutants which are characterized by their ability to selectively degradesteroids with or without 17-alkyl side chains of from 2 to 10 carbonatoms, inclusive, and accumulate predominantly the following compoundsin the fermentation beer:

3aα-H-4α-[3'-propanol]-7aβ-methylhexahydro-1,5-indandione hemiketal##STR1## hereinafter referred to as Compound I:

3aα-H-4α-[3'-propanal]-5α-hydroxy-7aβ-methylhexahydro-1-indanonehemiacetal ##STR2## hereinafter referred to as Compound II;

3aα-H-4α-[3'-propionicacid]-5α-hydroxy-7aβ-methylhexahydro-1-indanone-δ-lactone ##STR3##hereinafter referred to as Compound III: and,

3aα-H-4α-[3'-propanol]-5α-hydroxy-7aβ-methylhexahydro-1-indanone##STR4## hereinafter referred to as Compound IV.

These mutants can be obtained from sterol-degrading microorganisms ofthe following genera by using the mutation procedures disclosed hereinor other mutation procedures: Arthrobacter, Bacillus, brevibacterium,Corynebacterium, Mycobacterium, Nocardia, Protaminobacter, Serratia, andStreptomyces. A preferred genus is Mycobacterium. Exemplary species ofthis genus are M. phlei, M. smegmatis, M. rhodochrous, M. mucosum, M.fortuitum, and M. butyricum. Specifically exemplified herein is a novelmutant microorganism, Mycobacterium fortuitum, NRRL B-8128, which isused to selectively degrade steroids with or without 17-alkyl side chaincontaining from 2 to 10 carbon atoms, inclusive, to the compounds I, II,III and IV. Examples of suitable steroids are sitosterols, cholesterol,stigmasterol, campesterol, and like steroids with 17-alkyl side chainsof from 2 to 10 carbon atoms, inclusive, and androst-4-ene-3,17-dione,androsta-1,4-diene-3,17-dione, dehydroepiandrosterone, and testosterone.These steroids substrates can be in either the pure or crude form.

DETAILED DESCRIPTION OF THE INVENTION The Microorganisms

Mutants which are characterized by their ability to selectively degradesteroids, as defined above, and accumulate predominantly compoundsI,II,III and IV in the fermentation beer, can be obtained by mutatingsterol-degrading microorganisms of the following genera: Arthrobacter,Bacillus, Brevibacterium, Corynebacterium, Mycobacterium, Nocardia,Protaminobacter, Serratia, and Streptomyces. Mycobacterium fortuitum,ATCC 6842, has been mutated, as disclosed herein, to give a novellaboratory mutant microorganism. The 1974 ATCC Catalogue discloses thefollowing alongside the listing of ATCC 6842: "J. C. Cruz 2. Coldabscess. Acta Med. Rio de Janeiro 1:1 (1936). Medium 90 37C." M.fortuitum, ATCC 6842, degrades sterols non-selectively to smallmolecular weight compounds, e.g. CO₂ +H₂ O. Thus, this microorganism isnot suitable as a selective steroid degrader.

Mutation of M. fortuitum, ATCC 6842, using nitrosoguanidine has resultedin the production of a novel mutant which selectively degrades steroids,as defined herein, to accumulate predominantly compounds I, II, III andIV. This mutant microorganism of M. fortuitum has been given theaccession number NRRL B-8128, by the Northern Regional ResearchLaboratory, U.S. Department of Agriculture, Peoria, Ill., U.S.A., whereit has been deposited in the permanent collection. A subculture of thismicroorganism is freely available from this depository by request madethereto. It should be understood that the availability of the culturedoes not constitute a license to practice the subject invention inderogation of patent rights granted with the subject instrument bygovernmental action.

The morphology and drug sensitivities of M. fortuitum, NRRL B-8128, areindistinguishable from that of the parent M. fortuitum, ATCC 6842. BothM. fortuitum cultures are acid-fast non-motile, non-spore-formingbacilli belonging to the family Mycobacteriaceae of the orderActinomycetales. According to Runyon's classification (Runyon, E. H.1959 Med. Clin. North America 43: 273) it is a nonchromogenic group IVmycobacterium, i.e., it grows rapidly at low temperature to producenonpigmented colonies on relatively simple media.

M. fortuitum ATCC 6842 and M. fortuitum NRRL B-8128, are clearlydistinguishable in their action on steroid molecules. As disclosedabove, M. fortuitum ATCC 6842 is a non-selective degrader of steroids,whereas M. fortuitum NRRL B-8128 is a selective degrader. This propertyof M. fortuitum NRRL B-8128 makes it highly useful, as disclosed herein.

The mutation of M. fortuitum ATCC 6842 to give M. fortuitum NRRL B-8128was accomplished by the use of nitrosoguanidine. The details of theprocedure are described infra. Though mutation procedures are generallyknown in the art, there is no known art which teaches or even suggeststhe type of mutants, if any, which might be obtained by use of thesubject mutation procedure. Also, though the mutation and transformationprocedures, disclosed herein, are detailed for a Mycobacterium, itshould be understood that similar or equivalent procedures can be usedwith microorganisms of the other genera, as disclosed herein.

The Transformation Process

The selective transformation of the subject invention can be effected ina growing culture of M. fortuitum NRRL B-8128 by either adding theselected steroid substrate to the culture during the incubation period,or incorporating it in the nutrient medium prior to inoculation. Thesteriod can be added singly or in combination with another steroid. Thepreferred, but not limiting, range of concentration of the steroid inthe culture is about 0.1 to about 100 grams per liter. The culture isgrown in a nutrient medium containing a carbon source, for example, anassimilable carbohydrate, and a nitrogen source, for example, anassimilable nitrogen compound or proteinaceous material. Preferredcarbon sources include glucose, brown sugar, sucrose, glycerol, starch,cornstarch, lactose, dextrin, molasses, and the like. Preferred nitrogensources include cornsteep liquor, yeast, autolyzed brewer's yeast withmilk solids, soybean meal, cottonseed meal, cornmeal, milk solids,pancreatic digest of casein, fish meal, distillers' solids, animalpeptone liquors, meat and bone scraps, ammonium salts and the like.Combinations of these carbon and nitrogen sources can be usedadvantageously. Trace metals, for example, zinc, magnesium, manganese,cobalt, iron, and the like, need not be added to the fermentation mediasince tap water and unpurified ingredients are used as components of themedium prior to sterilization of the medium.

The transformation process can range from about 72 hours to 15 days. Theincubation temperature during the transformation process can range fromabout 25° C. to about 37° C., with 30° C. being preferred. The contentsof the transformation vessel are aerated with sterilized air andagitated to facilitate growth of the microorganism, and, thus, enhancethe effectiveness of the transformation process.

Upon completion of the transformation process, as evidenced by thinlayer chromatography using silica gel plates (E. Merck. Darmstadt) and asolvent system consisting of 2:3 (by volume) ethyl acetate-cyclohexane,the desired transformed steroids are recovered by means well known inthe art. For example, the fermentation (transformation) reactionmixture, including the fermentation liquor and cells, can be extractedwith a water-immiscible organic solvent for steroids. Suitable solventsare methylene chloride (preferred), chloroform, carbon tetrachloride,ethylene chloride, trichloroethylene, ether, amyl acetate, benzene andthe like.

Alternatively, the fermentation liquor and cells can be first separatedby conventional methods, e.g., filtration or centrifugation, and thenseparately extracted with suitable solvents. The cells can be extractedwith either water-miscible or water-immiscible solvents. Thefermentation liquor, freed of cells, can be extracted withwater-immiscible solvents.

The extracts can be filtered through diatomaceous earth and the filtratevacuum distilled to dryness. The resulting residue containing thedesired transformed steroids then can be dissolved in 10% chloroform inSkellysolve B and chromatographed on silica gel, using Skellysolve B(isomeric hexanes) and mixtures thereof with increasing amounts of ethylacetate as developing solvent. This procedure elutes compounds I, II andIII. Compound IV can be eluted with, for example, 5% methanol in ethylacetate. Crystalline products can be obtained by use of a solvent, forexample, ethyl acetate. The desired transformed steroids can also beobtained from the remaining supernatant upon evaporation of the solventin the supernatant.

Compounds I, II, III and IV, are useful as intermediates in the chemicalsynthesis of useful steroids. For example, the subject compounds can beconverted to starting material for the process disclosed in U.S. Pat.No. 3,880,884 which discloses a process for the total synthesis ofuseful 19-nor steroids. This conversion to starting material can be doneby procedures known in the art, e.g., by oxidation with chromic acid inacetic acid followed by treatment with acetic anhydride and sodiumacetate. See Biochem. 2: 1238-1243 and J.A.C.S. 85: 2135-2137.

The following examples are illustrative of the process and products ofthe subject invention but are not to be construed as limiting. Allpercentages are by weight and all solvent mixture proportions are byvolume unless otherwise noted.

EXAMPLE 1 Preparation of Mutant M, fortuitum NRRL B-8128 From M.fortuitum ATCC 6842 (a) Nitrosoquanidine Mutagenesis

Cells of M. fortuitum ATCC 6842 are grown at 28° C. in the followingsterile seed medium:

    ______________________________________                                        Nutrient Broth (Difco)                                                                              8      g/liter                                          Yeast Extract         1      g/liter                                          Sodium Propionate     0.5    g/liter                                          Distilled Water, q.s. 1      liter                                            ______________________________________                                    

The pH is adjusted to 7.0 with 1 N NaOH prior to sterilization at 121°C. for 20 minutes.

The cells are grown to a density of about 5×10⁸ per ml, pelleted bycentrifugation, and then washed with an equal volume of sterile 0.1 Msodium citrate, pH 5.6. Washed cells are resuspended in the same volumeof citrate buffer, a sample removed for titering (cell count), andnitrosoquanidine added to a final concentration of 50 μg/ml. The cellsuspension is incubated at 37° C. in a water bath for 30 minutes, afterwhich a sample is again removed for titering and the remaindercentrifuged down and washed with an equal volume of sterile 0.1 Mpotassium phosphate, pH 7.0. Finally, the cells are resuspended in asterile minimal salts medium, minus a carbon source, consisting of thefollowing:

    ______________________________________                                        NH.sub.4 NO.sub.3    1.0     g/liter                                          K.sub.2 HPO.sub.4    0.25    g/liter                                          MgSO.sub.4 . 7H.sub.2 O                                                                            0.25    g/liter                                          NaCl                 0.005   g/liter                                          FeSO.sub.4 . 7H.sub.2 O                                                                            0.001   g/liter                                          Distilled Water, q.s.                                                                              1       liter                                            ______________________________________                                    

The pH is adjusted to 7.0 with 1 N HCl prior to sterilization at 121° C.for 20 minutes. The cells are then plated out to select for mutants.

(b) Selection And Isolation Of Mutant M. fortuitum NRRL B-8128

Mutagenized cells, as described above, are diluted and spread ontoplates containing a medium consisting of the following (modified fromFraser and Jerrel. 1963. J. Biol. Chem. 205: 291-295):

    ______________________________________                                        Glycerol             10.0    g/liter                                          Na.sub.2 HPO.sub.4   8.4     g/liter                                          KH.sub.2 PO.sub.4    4.5     g/liter                                          NH.sub.4 Cl          2.0     g/liter                                          MgSO.sub.4 . 7H.sub.2 O                                                                            0.3     g/liter                                          FeCl.sub.3 . 6H.sub.2 O                                                                            0.05    g/liter                                          Distilled Water, q.s.                                                                              1       liter                                            ______________________________________                                    

Agar (15 g/liter) is added, and the medium is autoclaved at 121° C. for30 minutes and then poured into sterile Petri plates.

Growth on this medium eliminates most nutritional auxotrophs produced bythe mutagenesis procedure, e.g. cultures that require vitamins, growthfactors, etc. in order to grow on chemically defined medium areeliminated. After incubation at 28° C. for about 7 days, the resultingcolonies are replicated to test plates suitable for selecting mutantsand then back onto control plates containing the glycerol-based medium.The test plates are prepared as described by Peterson, G. E., H. L.Lewis and J. R. Davis. 1962. "Preparation of uniform dispersions ofcholesterol and other water-insoluble carbon sources in agar media." J.Lipid Research 3:275-276. The minimal salts medium in these plates is asdescribed above in section (a) of Example 1. Agar (15 g/liter), and anappropriate carbon source (1.0 g/liter), such as sitosterol orandrostenedione (AD), are added and the resulting suspension autoclavedfor 30 minutes at 121° C. The sterile, hot mixture is then poured into asterile blender vessel, blended for several minutes, and then pouredinto sterile Petri plates. Foaming tends to be a problem in thisprocedure but can be reduced by blending when the mixture is hot and byflaming the surface of the molten agar plates. In this manner uniformdispersions of water-insoluble carbon sources are obtained whichfacilitates the preparation of very homogenous but opaque agar plates.

Colonies which grew on the control plates, but not on test platescontaining AD as the sole carbon source, are purified by streaking ontonutrient agar plates. After growth at 28° C., individual clones arepicked from the nutrient agar plates with sterile toothpicks andretested by inoculating grided plates containing AD as the carbonsource. Purified isolates which still exhibit a phenotype different fromthe parental culture are then evaluated in shake flasks.

(c) Shake Flask Evaluation

Shake flasks (500 ml) contain 100 ml of biotransformation mediumconsisting of the following ingredients:

    ______________________________________                                        Glycerol             10.0   g/liter                                           Na.sub.2 HPO.sub.4   8.4    g/liter                                           KH.sub.2 PO.sub.4    4.5    g/liter                                           NH.sub.4 Cl          2.0    g/liter                                           MgSO.sub.4 . 7H.sub.2 O                                                                            0.3    g/liter                                           FeCl.sub.3 . 6H.sub.2 O                                                                            0.05   g/liter                                           Distilled Water, q.s.                                                                              1      liter                                             ______________________________________                                    

Soyflour (1 g/liter) is blended into the medium and then sitosterol (10g/liter) is also blended into the medium. After the flasks areautoclaved for 20 minutes at 121° C., they are cooled to 28° C. and theninoculated with 10 ml of seed growth prepared as follows:

The purified isolates from part (b) are grown on agar slants at 28° C. Aloop of cells taken from a slant is used to inoculate a 500-ml flaskcontaining 100 ml of sterile seed medium consisting of the followingingredients:

    ______________________________________                                        Nutrient Broth (Difco)                                                                              8      g/liter                                          Yeast Extract         1      g/liter                                          Glycerol              5      g/liter                                          Distilled Water, q.s. 1      liter                                            ______________________________________                                    

The pH is adjusted to 7.0 with 1 N NaOH prior to autoclaving the flasksat 121° C. for 20 minutes. The seed flasks are incubated at 28° C. for72 hours.

As disclosed above, 10 ml of seed growth is then used to inoculate each500-ml flask containing 100 ml of sterile transformation medium. Theflasks are then incubated at 28° C. to 30° C. on a rotary shaker andsampled at various intervals. Ten ml samples are removed and extractedby shaking with 3 volumes of methylene chloride. Portions of theextracts are analyzed by thin layer chromatography using silica gel andthe solvent system described above, i.e., 2:3 (by volume) ethylacetatecyclohexane, and by gas-liquid chromatography. Evidence of thepresence of compounds I, II, III and IV confirms the selectivedegradation of sitosterol by the novel mutant produced from the parentM. fortuitum ATCC 6842.

EXAMPLE 2 Transformation of Sitosterol to Compounds I, II, III and IV.

The medium used is the same as in Example 1c. This medium is sterilizedby heating 30 minutes at 121° C., whereupon it is cooled to 30° C. andthen inoculated with 10 parts of a seed culture of the mutantmycobacterium M. fortuitum NRRL B-8128, prepared as described in Example1 (c). The inoculated mixture is incubated at 30° C. for 336 hours withagitation to promote submerged growth. Upon completion of thisincubation period, thin layer chromatography analysis of thefermentation medium shows that compounds I, II, III and IV areaccumulated predominantly. Following incubation, the fermentation beeris extracted with methylene chloride. The extract is filtered throughdiatomaceous earth and the filtrate is vacuum distilled to dryness. Theresidue is taken up in 10% chloroform in Skellysolve B andchromatographed on silica gel, using Skellysolve B and mixtures thereofwith increasing amounts of ethyl acetate as developing solvent. Thisprocedure elutes compounds I, II and III. Compound IV is eluted from thecolumn with 5% methanol in ethyl acetate. The order of elution of thecompounds is I→II→III→IV. The R_(f) values for these compounds on thinlayer chromatography (tlc) is as follows (cyclohexane-ethyl acetate;3:2):

I=0.37

II=0.28

III=0.16

IV=0.05

Appropriate fractions, as determined by tlc, are pooled and evaporatedto dryness to afford good yields of compounds I, II, III and IV.

Upon recrystallization from ethyl acetate, compound I, a 1:1 mixture oftwo epimers, melts at 100°-112° C.;

compound II is an oil;

compound III melts at 122°-124° C.; and

compound IV melts at 148°-150° C.

Example 3

By substituting cholesterol for sitosterol in Example 2 there isaccumulated predominantly compounds I, II, III and IV.

EXAMPLE 4

By substituting stigmasterol in Example 2 for sitosterol there isaccumulated predominantly compounds I, II, III and IV.

EXAMPLE 5

By substituting campesterol for sitosterol in Example 2 there isaccumulated predominantly compounds I, II, III and IV.

EXAMPLE 6

By adding a combination of any of the steroids in Examples 2-5, inaddition to sitosterol, or in place of sitosterol, in Example 2 there isaccumulated predominantly compounds I, II, III and IV.

EXAMPLE 7

By substituting a sterol-degrading microorganism from the generaArthrobacter, Bacillus, Brevibacterium, Corynebacterium, Nocardia,Protaminobacter, Serratia, and Streptomyces, in Example 1 forMycobacterium fortuitum ATCC 6842 there are obtained mutantmicroorganisms which are characterized by their ability to selectivelydegrade steroids with or without a 17-alkyl side chain of from 2 to 10carbon atoms, inclusive, and accumulate predominantly compounds I, II,III and IV in the fermentation beer.

EXAMPLE 8

By substituting the mutants obtained in Example 7 for M. fortuitum NRRLB-8128 in Examples 2-6, there is accumulated predominantly compounds I,II, III and IV.

EXAMPLE 9

By substituting a sterol-degrading microorganism selected from the groupconsisting of Mycobacterium phlei, M. smegmatis, M. rhodochrous, M.mucosum, and M. butyricum for M. fortuitum ATCC 6842 in Example 1 thereare obtained mutant microorganisms which are characterized by theirability to selectively degrade steroids with or without a 17-alkyl sidechain of from 2 to 10 carbon atoms, inclusive, and accumulatepredominantly compounds I, II, III and IV in the fermentation beer.

EXAMPLE 10

By substituting the mutants obtained in Example 9 for M. fortuitum NRRLB-8128 in Examples 2-6, there is accumulated predominantly compounds I,II, III and IV.

EXAMPLE 11

By substituting a compound selected from the group consisting ofandrost-4-ene-3,17-dione, androsta-1,4-diene-3,17-dione,dehydroepiandrosterone, and testosterone for sitosterol in Example 2there is accumulated predominantly compounds I, II, III and IV.

EXAMPLE 12

By substituting a combination of two or more compounds selected from thegroup consisting of sitosterol, cholesterol, stigmasterol,androst-4-ene-3,17-dione, androsta-1,4-diene-3,17-dione,dehydroepiandrosterone, and testosterone for sitosterol in Example 2there is accumulated predominantly compounds I, II, III and IV.

EXAMPLE 13

By substituting the mutants obtained in Example 7 for M. fortuitum NRRLB-8128 in Examples 11 and 12, there is accumulated predominantlycompounds I, II, III and IV.

EXAMPLE 14

By substituting the mutants obtained in Example 9 for M. fortuitum NRRLB-8128 in Examples 11 and 12, there is accumulated predominantlycompounds I, II, III and IV.

What is claimed is:
 1. A biologically pure culture of mutantMycobacterium fortuitum NRRL B-8128.